www.elsevier.com/locate/brainres

Brain Research 1064

Research Report

The effect of low estrogen state on serotonin transporter function in mouse

hippocampus: A behavioral and electrochemical study

Paul P. Bertranda, Udeni T. Paranavitanea,b, Carolina Chavezb, Andrea Gogosb,c,

Margaret Jonesd, Maarten van den Buuseb,c,*

aDepartment of Physiology, University of Melbourne, Parkville VIC 3010, AustraliabBehavioural Neuroscience Laboratory, Mental Health Research Institute, Parkville VIC 3052, Australia

cDepartment of Pharmacology, University of Melbourne, Parkville VIC 3010, AustraliadPrince Henry’s Institute of Medical Research, Clayton VIC 3168, Australia

Accepted 11 October 2005

Available online 18 November 2005

Abstract

Defects in serotonergic transmission, including serotonin transporter (SERT) function, have been implicated in depression, anxiety

disorders and some aspects of schizophrenia. The sex steroid hormone estrogen is known to modulate functional SERT activity, but whether it

is up- or down-regulated is unclear. The aim of the present study was to examine the effect of a low estrogen state in mice on the behavioral

effect of drugs acting through the SERT, serotonin uptake kinetics and SERT density in the hippocampus. We compared control mice,

ovariectomized (OVX) C57BL/6J mice and aromatase knockout (ArKO) mice that are unable to produce estrogen. Fluoxetine treatment, but

not fenfluramine treatment, significantly increased prepulse inhibition (PPI), a measure of sensorimotor gating, in C57BL/6J mice. The effect

of fluoxetine was greater in OVX compared to sham-operated mice. In ArKO and J129 wild-type mice, fluoxetine increased PPI to the same

extent while fenfluramine increased PPI more in ArKO mice compared to controls. Measurement of the time-course for diffusion and

reuptake of exogenous serotonin in the CA3 region of the hippocampus showed that, in OVX mice, the fluoxetine-induced slowing of signal

decay after application of serotonin was enhanced when compared to sham-operated controls. Similarly, in ArKO mice, the effect of

fluoxetine was enhanced, suggesting that SERT function was greater than in J129 wild-type controls. Measurement of SERT density by [3H]-

citalopram autoradiography, revealed an 18% decrease in hippocampus of OVX mice compared to intact controls. SERT density was also

significantly reduced in nucleus accumbens (26%) but not in other regions, such as the raphe nuclei. Together, these results suggest that a low

estrogen state increases SERT activity in the hippocampus despite an apparent reduction in SERT density. The behavioral consequences of

these changes depend on the model of estrogen state used.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Serotonin

Keywords: Serotonin; Serotonin transporter; Estrogen; Hippocampus; Aromatase knockout (ArKO)

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.10.018

Abbreviations: ANOVA, analysis of variance; ArKO, aromatase

knockout mice; csf, cerebrospinal fluid; 5-HT, 5-hydroxytrytamine or

serotonin; OVX, ovariectomized; PPI, prepulse inhibition; SEM, standard

error of the mean; SERT, serotonin transporter

* Corresponding author. Behavioural Neuroscience Laboratory, Mental

Health Research Institute, 155 Oak Street, Parkville VIC 3052, Australia.

Fax: +61 3 93875061.

E-mail address: mvandenbuuse@mhri.edu.au (M. van den Buuse).

1. Introduction

Defects in serotonergic transmission, including serotonin

transporter (SERT) function, have been implicated in

depression, anxiety disorders and some aspects of schizo-

phrenia [18]. Levels of the sex steroid hormone, estrogen,

have been correlated with the severity of these diseases and

estrogen administration is known to change SERT expres-

sion and function, but the literature is unclear on the

(2005) 10 – 20

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–20 11

direction of the change. For example, some neurochemical

studies have found that in female rats in a low estrogen state

there is a decreased SERT gene expression and binding in

comparison to estrogen treatment groups [23]. In the same

species, an in vivo microdialysis study yielded results

consistent with less activity of SERTwhen circulating levels

of estrogen were low as compared to when estrogen levels

were high [21]. Similarly, Lu et al. showed in ovariecto-

mized female macaques that SERT binding and 5-HT

uptake were reduced as compared to hormone treated

animals [19]. In contrast, two recent studies found that in

ovariectomized rats or monkeys there was an up-regulation

of SERT gene expression as compared to animals chroni-

cally treated with estrogen receptor modulators [28,39].

SERT is crucial for terminating the serotonin signal and for

restricting its activity to the synaptic cleft. If estrogen

induces alterations in SERT function, it would have major

consequences for serotonergic activity in the brain and,

hence, neurochemical and behavioral mechanisms that

depend on serotonergic transmission.

The aim of the present project was to use functional

rather than neurochemical measures to look at the effect of

estrogen on SERT function. We used two main experimental

approaches, prepulse inhibition (PPI) of acoustic startle and

electrochemical detection of serotonin reuptake by SERT,

and two animal models of low estrogen state, ovariecto-

mized (OVX) mice and aromatase knockout mice (ArKO).

ArKO mice have been generated by targeted disruption of

the aromatase gene (CYP19) [8] which renders these

animals unable to produce estrogen. These functional

measures were compared to SERT density measurements

in hippocampus and other brain regions of OVX mice.

The first experimental approach used PPI of acoustic

startle to examine sensorimotor gating [11]. A startle reflex

is reduced when a strong stimulus is preceded by a weak

non-startling prestimulus [3]. PPI is controlled by several

central pathways including serotonergic input to the

hippocampus [15,34]. Serotonergic projections to the

hippocampus arise from the raphe nuclei [1,22,36,37] and

PPI is disrupted when these nuclei are selectively lesioned

[17]. Some psychiatric disorders, such as schizophrenia, are

characterized by deficient sensorimotor gating. Because PPI

depends on hippocampal serotonin function, this behavioral

test is a useful way to assess SERT function in the intact

animal. The second experimental approach involved assess-

ing 5-HT uptake kinetics with in vitro electrochemistry. The

clearance of exogenously applied 5-HT can be measured in

hippocampal slices using carbon fiber electrodes and high

speed chronoamperometry [4] and studies have shown that

serotonin clearance is faster in the dorsal raphe nucleus than

in the CA3 region [25]. Similarly, the long-term effects of

SERT inhibitors and the graded destruction of serotonergic

neurons cause a reduction in serotonin clearance [2,26].

Thus, comparisons between animals with altered SERT

activity may be detected as differences in the rate of

serotonin clearance. SERT density was assessed using [3H]-

citalopram autoradiography on sections of different regions

of the brain. The results suggest that a low estrogen state

increases SERT activity in the hippocampus, despite an

apparent reduction in SERT density. The behavioral con-

sequences of these changes depend on the model of estrogen

state used.

2. Experimental procedures

2.1. Animals

Female C57BL/6J mice (weight range 21–27 g), female

ArKO mice (weight range 23–30 g) and wild-type

littermates (weight range 21–27 g) were used. The ArKO

mice and their wild-type littermates were obtained from Dr.

Margaret Jones at Prince Henry’s Institute for Medical

Research (Clayton, Victoria, Australia). Heterozygous males

and females were bred to produce wild-type and homozy-

gous-null offspring. Mice were genotyped at weaning at

Prince Henry’s Institute for Medical Research using a PCR

based strategy [29]. While the genetic background of these

mice is a mix of C57BL/6J and J129 [8], the proportion of

J129 in the cohort of animals used for the present study was

found to be approximately 90% vs. the component of

C57BL/6J (M. Jones, personal communication). Therefore,

for the purpose of this study, the wild-type controls of ArKO

mice will be referred to as J129 wild-type controls.

Mice were housed at the Mental Health Research

Institute (Parkville, Victoria, Australia) in groups of 2–4

in standard mouse cages. C57BL/6J mice had free access to

standard pellet food and tap water. ArKO and J129 wild-

type mice were maintained on a soy-free mouse chow (Glen

Forest Stockfeeders, Australia) and tap water. The mice

were maintained on a 12/12 h light/dark cycle (lights on at

06:30, lights off at 18:30) at a constant temperature of 22

-C. The University of Melbourne Animal Experimentation

Ethics Committee approved all surgical techniques, treat-

ments and experimental protocols.

2.2. Surgery

A total of 15 C57BL/6J mice were ovariectomized at

approximately 12 weeks of age. The animals were anesthe-

tized (isoflurane/oxygen breathing mixture) and placed on a

heat pad maintained at 37 -C. A longitudinal midline incision

was made through the skin and blunt-tipped scissors were

used to separate the connective tissue and to cut through the

peritoneal wall. The ovaries were located and removed

bilaterally and the incision surgically stapled closed. In

sham-operated animals, all steps except removal of ovaries

were carried out. All mice were given a 5 mg/kg subcutane-

ous injection of the non-steroidal, anti-inflammatory analge-

sic carprofen (ZenecarpR, 50 mg/ml, Heriot AgVet, VIC,

Australia) to reduce pain and discomfort. Behavioural

experiments commenced 1–2 weeks after surgery.

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–2012

2.3. Drug treatment protocol

Each subgroup of C57BL/6J mice was randomly

injected with saline and 3 doses of one drug dissolved

in sterile saline. Using a randomized cross-over protocol,

every mouse from each group received all three drug

doses plus saline, with 3 or 4 days allowed between each

experiment to allow for the clearance of drugs. The first

group consisted of 7 OVX and 7 sham-operated mice and

received 3, 10 and 30 mg/kg of the selective SERT

inhibitor, fluoxetine ([T]-N-Methyl-g-[4-(trifluoromethyl)-

phenoxy] benzeneproanamine, Sigma-Aldrich, St. Louis,

Missouri, USA). The second group consisted of 8 OVX

and 8 sham-operated mice and received saline, 3, 10 or

30 mg/kg of the 5-HT releasing drug, fenfluramine (T-N-Ethyl-a-methyl-m-(trifluoromethyl) phenethylamine hydro-

chloride, Sigma-Aldrich). Fluoxetine or fenfluramine was

administered intraperitoneally, 20 min prior to the

commencement of the PPI session. Because of limited

availability of ArKO mice, these animals and their J129

wild-type littermates received only saline, 30 mg/kg of

fluoxetine or 30 mg/kg of fenfluramine in a randomized,

cross-over protocol.

2.4. Prepulse inhibition

Prepulse inhibition (PPI) experiments used a four-unit

automated SR-LAB startle system (San Diego Instruments,

CA, USA) that delivered startle stimuli and recorded

responses to a computer in an adjacent room. Mice were

individually placed into a transparent cylinder on a platform

with a piezoelectric transducer to detect motion. Animals

acclimatized for 5 min before the start of the PPI session.

During the session, animals were delivered a constant

background white noise of 70 decibels (dB) intensity

through speakers in the ceiling of the box.

The PPI session was approximately 50 min in duration

and consisted of 100 trials (pulses) [35,38]. It commenced

and ended with a group of 10 40 ms 115 dB startle-pulse

alone trials. Between these first and last groups of startle-

pulse alone trials, 80 more trials were delivered with a

variable inter-trial interval of 25 s average. These 80 trials

randomly consisted of 20 startle-pulse alone trials and 50

prepulse + startle-pulse trials. Prepulse trials consisted of

an 115 dB startle-pulse stimulus preceded 100 ms before

by a 20 ms prepulse of either 2, 4, 8, 12 or 16 dB (PP2,

PP4, PP8, PP12, PP16) over the 70 dB background (i.e.,

72, 74, 78, 82, 86 dB prepulses). In addition, there were

10 Fno stim_ trials to detect non-specific movement

artifacts. Drug effects on startle amplitude were assessed

using the 40 startle-pulse trials that were recorded

throughout the session. The percentage of PPI was

calculated as the difference of the magnitude of the startle

response to pulse-alone trials minus that to prepulse trials,

divided by the response to pulse-alone trials, expressed as

percentage [35,38].

2.5. Chronoamperometry

At least 3 days after the last behavioral experiments, mice

were deeply anesthetized with pentobarbitone sodium

(Nembutal, Rhone Merieux Australia Pty Ltd., QLD,

Australia) and decapitated. The brain was rapidly removed,

cut in the midsagittal plane and immersed in ice-cold

artificial cerebrospinal fluid (aCSF, composition in mM;

NaCl 126, NaHCO3 25, d-Glucose 11, KCl 2.5, CaCl2 2.4,

MgCl2 1.2, NaH2PO4 1.2) that was bubbled with 95% O2/

5% CO2. The initial solution also contained a high

magnesium concentration (7 mM). A 300 Am transverse

section of the brain was cut using a vibroslicer (Model

MA752, Campden Instruments Ltd., Leicestershire, UK)

then placed in high magnesium aCSF at room temperature

(22.0 T 3 -C) and gently heated to 37 -C for 1 h. One brain

slice was placed in an organ bath of 3 ml volume and held in

place with a metal mesh. The slice was superfused at 3 ml/

min with 35 -C aCSF of normal magnesium concentration

and allowed to equilibrate for 15–20 min. All electrochem-

ical experiments were conducted at 35 -C.A single 7 Am diameter carbon fiber was inserted into a

borosilicate glass micropipette (Harvard Apparatus, SDR

Clinical Technology, NSW, Australia) and pulled with a

micropipette puller (Narishige, Tokyo, Japan). A pellet of

Woods metal (Amac Alloys, VIC, Australia) was inserted

into the open end of the micropipette and copper wire (0.8

mm diameter, Oz wire, Australia) was inserted behind it.

The Woods metal was then gently heated to establish a

stable electrical connection between the copper and carbon

fiber. The exposed carbon fiber was trimmed to 100–200

Am in length. To improve the specificity of the electrodes,

the tips were cleaned in 5% acetone, then coated with

NafionR 117 solution (15 min, 3% solution, Fluka Chemika,

Switzerland). Nafion is a perfluorosulfonated ion exchange

resin that excludes negatively charged molecules such as

monoamine metabolites and ascorbic acid [10].

Serotonin was dissolved in distilled water (5 AM, 5-

hydroxytryptamine creatinine sulfate, Sigma-Aldrich) and

placed in a pipette which was attached to a pressure ejection

system (Picospritzer III, Parker Instrumentation, VIC, Aus-

tralia). This concentration of 5-HT is high enough to saturate

all of the SERT high affinity binding sites in the area local to

the pipette. The same solution was used to calibrate the

carbon fiber electrode at the beginning of each experiment

and to examine clearance of 5-HT from the hippocampus.

An oxidizing potential of +400 mV was applied to the

carbon fiber for 15–60 s with an electrochemistry amplifier

(NPI electronics VA-10, ALA Scientific instruments, NY,

USA) and 5-HT was pressure ejected onto the exposed

carbon fiber (50–150 ms pulse duration, 10 p.s.i.). The

electrode was returned to a holding potential of 0 mV

between applications of 5-HT. Serotonin oxidation signals

were digitized between 1 and 5 kHz (Digidata 1200)

recorded to a personal computer and analyzed with

Axoscope 9 (All from Axon Instruments, CA, USA).

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–20 13

The hippocampus was visualized at 40� magnification

with a dissecting microscope. The carbon fiber electrode and

the 5-HTcontaining pipette were aligned tip to tip in the CA3

region of the hippocampus using mechanical micromanipu-

lators. This area was chosen because the noradrenaline

transporter is not a confounding factor in this area [5]. The

position of the electrode and micropipette was adjusted until

the greatest amplitude oxidation current to application of

serotonin was found—this amplitude corresponded to be-

tween 1 and 5 AM 5-HT. Control 5-HT oxidation currents

were recorded once every 5min for 25–35min (6–8 repeats).

Fluoxetine (1 nM) was superfused onto the bath and allowed

to equilibrate for 15–20 min before 5-HT oxidation currents

were again recorded every 5 min (6–8 repeats). In control

experiments, this concentration of fluoxetine was the

minimum needed to cause a clear reduction in 5-HT

clearance. Fluoxetine was allowed to wash out of the bath

for 15–20 min before recovery recordings were made (6–

8 repeats).

2.6. SERT density

For this experiment, another group of C57BL/6 mice was

ovariectomized or sham-operated as described above. There

were no ArKO mice available for SERT density measure-

ments. Four weeks after surgery, animals were killed by

decapitation and their brains were removed and frozen over

dry ice. Sections of the brain were cut at 14 Am on a cryostat

(Leica CM18-50, Leica Microsystems Nussloch GmbH,

Germany) at �20 -C and thaw-mounted onto gelatinized

microscope slides. Regions selected were at the level of

nucleus accumbens (bregma 1.18 to 0.86), dorsal hippo-

campus (bregma �1.82 mm to �2.18 mm) and raphe nuclei

(bregma �4.36 mm to �4.72 mm) [9]. Brain sections were

stored at �80 -C until use.

Sections were allowed to thaw, washed for 15 min in

Tris–HCl buffer (120 mM NaCl (Asia Pacific Specialty

Chemicals; Seven Hills, NSW, Australia), 50 mM Tris–

HCl (Sigma-Aldrich), pH 7.4) at room temperature and

dried with a stream of cool air. For specific total binding,

Tris–HCl buffer was supplemented with 2 nM [3H]-

citalopram (84.0 Ci/mmol; Amersham Bioscience; Castle

Hill, NSW, Australia). Non-specific binding was deter-

mined by adding 10 AM of the selective SERT inhibitor,

fluoxetine (Sigma-Aldrich) in addition to 2 nM [3H]-

citalopram. Fifty microliters of the 2 nM solution was

dispensed on top of each tissue section and allowed to

incubate for 1 h. Sections were then washed three times (1,

10 and 10 min, respectively) in ice-cold Tris–HCl buffer.

Once dry, sections were partially fixed with 4% parafor-

maldehyde vapor overnight. Fixed sections were apposed to

a BAS-TR2025 phosphoimaging plate (Fuji Imaging

Plates, Berthold Australia Pty. Ltd., Bundoora, Victoria,

Australia) for 10 days at room temperature in a cassette

together with autoradiographic [3H]-micro-scales (Amer-

sham Bioscience, Buckinghamshire, England).

Autoradiographic images on plates were scanned from

the phosphoimaging plate and images were retrieved for

analysis using AIS image analysis software (Analytical

Imaging Station, Imaging Research Inc., Ontario, Canada).

A standard curve was calculated according to the standard

tritium microscales, allowing conversion of photo-stimulat-

ed luminescence (psl) to desintegration units per minute per

milligram (dpm/mg) estimated tissue equivalent (ETE).

Images were then quantified against the standard curve.

The density of specific SERT binding was calculated by

subtracting the density from the non-specific samples from

that of the total binding samples. The numbers were then

converted from dpm/mg ETE to fmol/mg ETE using the

specific activity of the ligand and a decay factor.

2.7. Data analysis

All data were expressed as mean T standard error of the

mean (SEM). A one-way analysis of variance (ANOVA)

(SYSTAT 9, SPSS, Illinois, USA) followed by Bonferroni-

corrected t test was used to analyze body weight and uterus

weight data and SERT density data.

PPI data were analyzed using two- and three-way

ANOVA with repeated measures where appropriate

(SYSTAT). Data for startle amplitude and PPI from sham-

operated and OVX mice or J129 wild-type and ArKO mice

were compared with group (sham-operated and OVX or

J129 wild-type and ArKO) as a between-group factor and

dose (of either fenfluramine or fluoxetine in C57BL/6J) or

treatment (saline, fenfluramine or fluoxetine in ArKO mice)

as within-animal factor. An additional within-animal factor

was prepulse intensity for PPI analysis. Because the main

effect of prepulse intensity was always significant, it will not

be presented here in detail. Where appropriate, pair-wise

ANOVAs were used to further analyze the effect of

treatment or dose between the groups. Differences were

considered significant if P < 0.05.

Differences in electrochemical signals between control

and fluoxetine periods for a single brain slice were analyzed

by comparing the amplitude of the oxidation current and the

times for 50% and 90% decay to occur. Decay times were

calculated by taking the peak amplitude of the average trace

(3–6 repeats) for the control or fluoxetine period, and

finding the times at which the amplitude had fallen to one-

half peak amplitude and to one-tenth peak amplitude. The

absolute time course of the 5-HT induced current was

dependent on the placement of the electrode and the pipette.

To reduce this source of variability, the percent of control

change was calculated and used for all further analyses.

The close proximity of the electrode and 5-HT containing

pipette meant that the initial decay phase of the current (50%

decay time) was due to diffusion of 5-HT away from the

electrode while the effects of the SERT in the surrounding

tissue would be detected primarily at the 90% decay times.

Similarly, the amplitude of the current was primarily

controlled by the concentration of 5-HT in the pipette.

Table 2

Body weights, uterus weights, average startle amplitudes and average

percentage prepulse inhibition (PPI) of sham-operated and ovariectomized

(OVX) C57BL/6J mice treated with fenfluramine

Group Sham (n = 7) OVX (n = 7)

Body weight (g) 21.7 T 0.3 25.1 T 1.2*

Uterus weight (mg) 57.2 T 4.9 16.3 T 0.5*

Uterus/body weight ratio 2.64 T 0.24 0.65 T 0.21*

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–2014

When near-maximal responses were recorded, as they were

in here, fluoxetine would not be expected to enhance them.

The electrochemical data were analyzed using an

ANOVA (SYSTAT). Comparisons were made for the effects

of fluoxetine in Sham vs. OVX mice and for the effects of

fluoxetine in J129 wild-type vs. ArKO mice. Differences

were considered significant if P < 0.05.

Average startle amplitude

Saline 107 T 13 146 T 24

Fenfluramine 3 mg/kg 132 T 13 189 T 38

Fenfluramine 10 mg/kg 160 T 14 282 T 80

Fenfluramine 30 mg/kg 122 T 20 221 T 32

Average %PPI

Saline 48.5 T 2.6 42.1 T 2.6

Fenfluramine 3 mg/kg 50.1 T 4.8 46.2 T 5.2

Fenfluramine 10 mg/kg 54.6 T 3.3 39.6 T 6.4

Fenfluramine 30 mg/kg 50.7 T 3.7 49.4 T 5.3

* P < 0.05 compared with sham-operated mice (t test). For further

statistical comparison, see text.

3. Results

Body weights and uterus weights at the time of sacrifice

were used to confirm the estrogen state of the animals used in

PPI and uptake kinetics experiments (Tables 1–3). Body

weight was significantly higher in one group of OVX mice

vs. their respective sham-operated controls (Tables 1 and 2).

Similarly, ArKO mice had a significantly higher body weight

compared to J129 wild-type mice (Table 3). Uterus weight

was markedly reduced in all OVX mice compared to sham-

operated C57BL/6J mice and, consequently, the uterus

weight/body weight ratio was significantly reduced (Tables

1 and 2). Uterus weight and uterus/body weight ratio were

similarly lower in ArKOmice than in J129 wild-type controls

(Table 3).

3.1. Effect of fluoxetine or fenfluramine on PPI in C57BL/6J

mice

Treatment with fluoxetine significantly increased startle

amplitudes (main effect of dose F(3,42) = 17.9, P < 0.001).

However, ovariectomy did not influence startle amplitude

(Table 1). Treatment with fluoxetine caused an increase of

PPI (main effect of dose F(3,42) = 4.2, P = 0.011) and this

effect was greater for lower prepulse intensities (interaction

of prepulse and dose F(12,168) = 7.4, P < 0.001). Although

Table 1

Body weights, uterus weights, average startle amplitudes and average

percentage prepulse inhibition (PPI) of sham-operated and ovariectomized

(OVX) C57BL/6J mice treated with fluoxetine

Group Sham (n = 7) OVX (n = 7)

Body weight (g) 25.5 T 0.6 24.5 T 0.6

Uterus weight (mg) 86.2 T 7.7 11.0 T 1.3*

Uterus/body weight ratio 3.38 T 0.29 0.45 T 0.05*

Average startle amplitude

Saline 140 T 16 138 T 17

Fluoxetine 3 mg/kg 130 T 16 149 T 19

Fluoxetine 10 mg/kg 192 T 22 180 T 21

Fluoxetine 30 mg/kg 208 T 30 215 T 16

Average %PPI

Saline 47.2 T 2.9 45.2 T 3.7

Fluoxetine 3 mg/kg 49.7 T 4.7 46.2 T 2.6

Fluoxetine 10 mg/kg 54.4 T 4.2 54.8 T 3.0

Fluoxetine 30 mg/kg 58.8 T 3.2 59.8 T 3.2

* P < 0.05 compared with sham-operated mice (t test). For further

statistical comparison, see text.

there was no main effect of Group (Table 1), the prepulse-

dependent effect of fluoxetine on PPI was significantly

greater in OVX mice than in sham-operated controls

(interaction of prepulse � dose � group F(12,168) =

10.1, P < 0.001) (Fig. 1). Treatment with fenfluramine

significantly increased startle amplitudes (main effect of

dose F(3,36) = 4.9, P = 0.006). However, as with fluoxetine

treatment, ovariectomy did not influence startle amplitude

(Table 2). Fenfluramine treatment had no effect on PPI in

either sham-operated or OVX mice (Fig. 2).

3.2. Effect of fluoxetine and fenfluramine on PPI in ArKO

mice

Analysis of combined data obtained after saline, fluox-

etine and fenfluramine treatment revealed a significant main

Table 3

Body weights, uterus weights, average startle amplitudes and average

percentage prepulse inhibition (PPI) of J129 wild-type controls and ArKO

mice

Group J129 wild-type (n = 8) ArKO (n = 8)

Body weight (g) 24.3 T 0.8 26.8 T 0.8*

Uterus weight (mg) 151.3 T 24.3 10.6 T 1.7*

Uterus/body weight ratio 6.38 T 1.13 0.39 T 0.06*

Average startle amplitude

Saline 174 T 47 222 T 52

Fluoxetine 30 mg/kg 198 T 36 338 T 51

Fenfluramine 30 mg/kg 168 T 46 270 T 54

Average %PPI

Saline 63.6 T 4.6 50.3 T 4.7

Fluoxetine 30 mg/kg 65.1 T 3.5 65.1 T 3.5

Fenfluramine 30 mg/kg 68.3 T 2.0 70.8 T 3.2

* P < 0.05 compared with wild-type J129 mice (t test). For further

statistical comparison, see text.

Fig. 2. Prepulse inhibition (PPI) of startle of C57BL/6J mice that were

either sham-operated (Sham, top panels) or ovariectomized (OVX, bottom

panels). The mice were treated with either saline (SAL) or 3, 10 or 30 mg/

kg of fenfluramine. Data are mean T SEM of 8 mice per group. For

statistical comparison, see text.

Fig. 1. Prepulse inhibition (PPI) of startle of C57BL/6J mice that were

either sham-operated (Sham, top panels) or ovariectomized (OVX, bottom

panels). The mice were treated with either saline (SAL) or 3, 10 or 30 mg/

kg of fluoxetine. Data are mean T SEM of 7 mice per group. For statistical

comparison, see text.

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–20 15

effect of treatment (F(2,28) = 7.0, P = 0.003). Further

pairwise analysis showed that fluoxetine treatment caused a

significant increase in startle amplitudes (F(1,17) = 15.2, P =

0.001) to the same extent in both genotype groups; however,

there was no significant effect of fenfluramine treatment on

startle amplitudes (Table 3). Analysis of combined data also

showed a significant overall treatment effect on PPI (main

effect of treatment:F(2,28) = 7.7,P = 0.002).While there was

no overall effect of genotype, the treatment effect showed a

trend to depend on the genotype (interaction of treatment and

group F(2,28) = 3.0, P = 0.066). Pairwise treatment analysis

showed that fluoxetine treatment induced a small, but

significant increase in PPI, particularly at higher prepulse

intensities (main effect of treatment F(1,14) = 5.1, P = 0.039;

interaction of treatment and prepulse F(4,56) = 2.8, P =

0.035). However, the effect of fluoxetine did not differ

between ArKO mice and J129 wild-type controls (Fig. 3). In

contrast, fenfluramine treatment markedly increased PPI

(main effect of treatment F(1,14) = 13.5, P = 0.003) and

this effect was significantly greater in ArKO mice than in

J129 wild-type controls (interaction of treatment and group

F(1,14) = 5.6, P = 0.033) (Fig. 3).

3.3. Serotonin uptake kinetics

Oxidation currents induced by pressure application of 5-

HT (5 AM) had a fast rising phase. The average time to peak

was 135 T 10 ms with a peak current of 54 T 6 nA (n = 49).

This peak current corresponds to almost 5 AM of 5-HT. The

Fig. 3. Prepulse inhibition (PPI) of startle of ArKO mice and their wild-type

(WT) controls that were treated with either saline (Sal), 30 mg/kg of

fluoxetine (Fluox) or 30 mg/kg of fenfluramine (Fen). Data are mean T

SEM of 8 mice per group. For statistical comparison, see text.

Fig. 5. Representative traces showing serotonin-induced oxidation current

(normalized) in the CA3 region of the hippocampus of female J129 wild-

type (WT) control mice (A) and female Aromatase knockout (ArKO) mice

(B). The lower trace for each panel is control and the upper trace is in the

presence of fluoxetine (1 nM). (C) Histogram with group data (mean T

SEM) for 7 mice per group. The effect of fluoxetine as a percent of control

is shown for times to 50% and 90% decay. ArKO mice showed a

significantly larger increase in 90% decay time than did the J129 wild-type

controls (*P < 0.05).

Fig. 4. Representative traces showing serotonin-induced oxidation current

(normalized) in the CA3 region of the hippocampus of female SHAM-

operated control mice (A) and female OVX mice (B). The lower trace for

each panel is control and the upper trace is in the presence of fluoxetine (1

nM). (C) Histogram with group data (mean T SEM) for 13 mice per group.

The effect of fluoxetine as a percent of control is shown for times to 50%

and 90% decay. OVX mice showed a significantly larger increase in 90%

decay time than did the SHAM controls (*P < 0.05).

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–2016

decay phase was composed of a fast decay and a slower Ffoot_at the end of the response; it was this later phase that was

affected by fluoxetine (e.g., Fig. 4A). On average, the times to

50% and 90% decay were 410 T 50 ms and 4190 T 770 ms,

respectively (n = 49). In order to reduce variability, all data

were normalized as a percent of (prefluoxetine) control.

In 13 sham-operated C57BL/6J mice, addition of

fluoxetine to the recording bath caused a small increase in

the time for 50% decay (123 T 14% of control, n = 13) and

in the time for 90% decay (121 T 8 of control). In C57BL/6J

OVX mice, this increase was similar for the time for 50%

decay (118 T 12% of control) but was significantly

prolonged for the time for 90% decay (183 T 24% of

control) (Fig. 4).

In J129 wild-type controls, addition of fluoxetine to

the recording bath caused a small increase in the time for

50% decay (115 T 10% of control, n = 7) and in the time

for 90% decay (119 T 6 of control). In ArKO mice, the

time for 50% decay was non-significantly increased to

142 T 35% of control while, however, the time for 90%

decay was significantly increased to 163 T 15% of control

(Fig. 5).

3.4. SERT density

Similar to the mice in the PPI and uptake kinetics

experiments, OVX mice used for SERT binding density

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–20 17

had significantly lower uterus weights either as absolute

values (24.9 T 3.6 mg vs. 84.8 T 8.8 mg in controls) or

as ratio of body weight (1.0 T 0.2 mg/g vs. 3.7 T 0.4

mg/g in controls). The highest binding occurred in the

lateral septum, followed by the DRN, MRN, hippocam-

pus and nucleus accumbens (core), respectively. The

lowest degree of binding occurred in the cingulate cortex.

As depicted in Fig. 6, SERT density was significantly

lower in OVX mice than in sham-operated controls in the

hippocampus (18% lower) and in the nucleus accumbens

(26% lower), but not in the DRN, MRN, lateral septum

and cingulate cortex.

Fig. 6. Mean T SEM SERT binding density as measured with specific [3H]-

citalopram autoradiography in sections of nucleus accumbens, cingulate

cortex, lateral septum, hippocampus, dorsal raphe nucleus (DRN) and

median raphe nucleus (MRN). SERT binding was significantly lower in

OVX mice than in sham-operated controls in nucleus accumbens and in

hippocampus (*P < 0.05).

4. Discussion

The main finding of this study was that a low estrogen

state increases SERT function, measured as serotonin uptake

kinetics, regardless of the model of estrogen state used. In

contrast, in PPI experiments, there were differential effects

of drugs acting on or through the SERT depending on the

model of low estrogen state used. SERT binding density was

significantly lower in hippocampus of OVX mice than

controls.

4.1. Serotonin uptake kinetics

We used high speed chronoamperometric electrochemi-

cal techniques to detect real time changes in serotonin

concentrations allowing us to determine its clearance from

the extracellular space [4]. Chronoamperometry has not

previously been used to assess the functional consequences

of low estrogen levels on serotonergic transmission. As a

measure of SERT activity, we assessed the time for 50% and

90% decay of the serotonin oxidation signal from its peak

amplitude and the ability of fluoxetine to increase these

parameters. Fluoxetine binds to the SERT and blocks the

reuptake of serotonin into the synaptic terminal [30]. In

vivo, the ensuing increase in extracellular serotonin in the

CA3 region may activate postsynaptic receptors which

results in a behavioral change. In vitro, acute fluoxetine

application blocked uptake of exogenously applied seroto-

nin which resulted in lengthening of the chronoampero-

metric decay curve [4]. A reduced effect of fluoxetine,

particularly on 90% decay time, would mean a lower SERT

activity under control conditions. Conversely, an increased

effect of fluoxetine would reflect a higher basal SERT

activity. In our experiments, we observed an enhanced in

vitro effect of fluoxetine (i.e., higher SERT density or

activity) in OVX and ArKO mice. This suggests that a low

estrogen state, regardless of how it was produced, increased

SERT function. It should be noted that while 5-HT was at a

saturating concentration, fluoxetine was not. Thus, incom-

plete block of SERT by fluoxetine, especially in ArKO or

OVX mice, may have contributed to the results.

There is considerable discrepancy in the literature as to

the effect of estrogen on SERT function or density. Some

neurochemical studies found that SERT gene expression and

binding [23] or function [21] are reduced in a low estrogen

state. Similarly, Lu et al. [19] showed that SERT binding

and 5-HT uptake were less in ovariectomized than in

hormone treated female macaques [19]. It is worth noting

that, in these studies, estrogen was administered acutely [23]

and SERT function was measured in the mediobasal

hypothalamus [19,21]. In contrast, other studies have found

that ovariectomized rats or monkeys showed up-regulation

of SERT gene expression [28,39] and density of SERT

binding sites [24] in comparison to animals chronically

treated with estrogen or selective estrogen receptor modu-

lators. In our OVX mice, autoradiographic density of SERT

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–2018

was reduced in hippocampus and nucleus accumbens,

consistent with a reduction in SERT gene expression or

numbers in the low estrogen state. These data are at odds

with our chronoamperometry results (discussed above). It is

not clear how reduced SERT binding sites could cause a

relatively greater capacity for serotonin reuptake. Further

chronoamperometry experiments would be needed in

control and in OVX mice to determine whether these

conditions cause a change in the maximal velocity of SERT

transport or the affinity for 5-HT.

4.2. Prepulse inhibition

Administration of fluoxetine caused an increase in startle

amplitude and PPI. Ovariectomy enhanced the effect of

fluoxetine on PPI, but not startle amplitude. The effect of

fluoxetine on startle and PPI has not previously been tested

in mice. Data in rats suggest that an increase in extracellular

serotonin levels, as a result of fluoxetine treatment, does not

affect PPI [20]. The difference between this study and our

results could be because of species differences or due to the

fact that we used female mice vs. male rats.

The mechanism of action of the ovariectomy-induced

enhancement of fluoxetine action on PPI remains unclear. It

has been shown that serotonin levels are decreased in the

hippocampus of OVX mice [14]. This could be caused by

two distinct mechanisms. First, reduced levels could be the

result of enhanced release. In this case, the effect of

fluoxetine in our experiments to increase PPI may be

enhanced in OVX mice because of an already increased

serotonin release in the low estrogen state. Second, the

reduced 5-HT levels in OVX mice could be due to increased

SERT activity as an up-regulation of SERT gene expression

[28,39] or density of SERT binding sites [24]. This is inline

with our chronoamperometry results which showed an

increased SERT function in OVX mice. In this case,

blockade of the SERT by fluoxetine would then cause a

relatively greater increase in extracellular serotonin levels in

OVX mice than in sham-operated controls and, consequent-

ly, a more marked behavioral effect, as was seen in our PPI

experiments.

It is harder to reconcile our findings with those that

show that ovariectomy causes a decrease in SERT density.

It was found that ovariectomy reduced SERT gene

expression in the brain, particularly in the dorsal raphe

nucleus and septum [23,33], as compared to acutely

ovariectomized rats injected with estrogen. This is consis-

tent with microdialysis experiments that showed that, in

diestrus, when circulating levels of estrogen are low, acute

treatment with fluoxetine caused a markedly smaller

increase in extracellular serotonin levels when compared

to estrus, when circulating levels of estrogen are high [21].

If, in our experiments, a loss of estrogen by ovariectomy

caused lower levels or activity of SERT, then it is difficult

to explain why there was an enhancement of fluoxetine

action on PPI. Thus, a decrease in SERT is consistent with

our autoradiography results, but not our chronoamperom-

etry or PPI results.

The administration of the serotonin releasing drug

fenfluramine, produced an increase in startle amplitude in

C57BL/6J mice, showing fenfluramine was active in the

brain at this dose. Nevertheless, fenfluramine treatment did

not significantly influence PPI in C57BL/6J mice. While the

effect of fenfluramine on PPI in mice has not previously

been examined, Dulawa and Geyer [6] reported that another

serotonin releasing drug, MDMA ((+)3,4-methylenedioxy-

N-methylamphetamine), had no effect on the startle

response in C57BL/6J mice but that PPI was decreased. It

may be that MDMA was having an effect on PPI that was

not related to its actions on the serotonergic system, but

rather by its action on central dopaminergic activity [6].

Fenfluramine is a substrate type releaser that initially binds

to the SERT and is transported into the nerve terminal [30].

There it promotes non-exocytotic release of serotonin by a

carrier-mediated exchange process [30]. It would appear that

these mechanisms are not altered in OVX C57BL/6J mice. It

is unclear why fluoxetine treatment increased PPI in sham-

operated and OVX C57BL/6 mice, whereas fenfluramine

treatment had no effect. Further experiments assessing

SERT gene expression and SERT binding in the brain are

needed to investigate this apparent discrepancy.

Fluoxetine administration caused an increase in startle

amplitudes and PPI in J129 wild-type controls and ArKO

mice; however, in contrast to the effect of fluoxetine in

C57BL/6J mice, there were no differences in the extent of

this effect between the groups. Treatment with fenfluramine,

which did not influence PPI in C57BL/6J mice, increased

PPI in J129 wild-type controls and ArKO mice. Moreover,

the effect of fenfluramine was greater in the ArKO mice

than in their controls.

4.3. Differences in models of estrogen state

There were two main differences between C57BL/6J

sham-operated and OVX mice on the one hand and J129

wild-type and ArKO mice on the other. First, the genetic

background may have played a role. Numerous studies have

shown mouse strain differences in behavior, including PPI

(for references, see [12,27]). This could explain why sham-

operated C57BL/6 mice differ from J129 wild-type mice

with respect to their responsiveness to fluoxetine and

fenfluramine. It would then be of particular interest to study

the effect of ovariectomy in J129 wild-type controls to see if

a similar enhancement of the effect of fenfluramine will

occur as seen in ArKO mice or if an enhancement of the

effect of fluoxetine would occur, similar to that seen in OVX

C57BL/6 mice. Furthermore, measurement of SERT density

would have to be done in ArKO mice.

The other main difference between the two models is the

way the low estrogen state is achieved. Ovariectomy will

remove one major source of circulating sex steroids;

however, this procedure will not inhibit production of

P.P. Bertrand et al. / Brain Research 1064 (2005) 10–20 19

estrogen elsewhere in the body, in particular the brain [32].

It is becoming clear that, in the brain, steroid hormones are

synthesized and may act locally to influence behavior in a

paracrine fashion [31]. Unlike ovariectomy, targeted dis-

ruption of the CYP19 gene in ArKO mice will also remove

components of this brain steroid production. This may

explain some of the differences in behavioral effects

between the low estrogen state achieved by ovariectomy

vs. disruption of the CYP19 gene.

It is important to note that ArKO mice not only show

reduced estrogen production but, as a consequence of the

mutation, also high circulating testosterone levels [8].

Estrogen is produced by conversion of testosterone by the

aromatase enzyme and blockade of this conversion may be

the cause of a Fbuild-up_ of testosterone [8]. It is possible

that high testosterone levels in the ArKO mice are

involved in the differential effects seen on the effect of

fluoxetine and fenfluramine on PPI [7], as we have shown

for the effect of the 5-HT1A receptor agonist, 8-OH-DPAT

[13]. Finally, as with other animal models using knockout

strategies, the deletion of the aromatase gene could have

effects throughout development and may trigger compen-

satory changes in responsiveness to drugs, such a

fluoxetine and fenfluramine.

Follow-up experiments should be aimed at other manip-

ulations of estrogen levels, particularly by following the

normal variation of these levels during the estrus cycle.

However, other hormone levels also change during this

cycle and therefore the present study was focused on just

estrogen.

In conclusion, we observed that low estrogen state,

induced either by ovariectomy or in ArKO mice,

increased SERT function as measured by chronoamper-

ometry in hippocampal slices. SERT binding density was

reduced in hippocampus of OVX mice, which is at odds

with the increased SERT kinetics. Interestingly, the

behavioral responses to drugs acting on or through the

SERT depended on how the low estrogen state was

induced. Considering that sensory motor gating is

disrupted in schizophrenia and other mental illnesses,

our experiments on the effect of low estrogen state may

provide further insight into the possible beneficial effects

of estrogen as adjuncts to these treatments [16].

Acknowledgments

This work was supported by the National Health and

Medical Research Council, Australia. Dr. M. van den

Buuse was supported by the Griffith Senior Research

Fellowship of the University of Melbourne. Dr. M. Jones

was supported by a U.S. Public Health Service Grant

(R37 AG-08174). The Mental Health Research Institute is

a Stanley Research Centre supported by the Stanley

Medical Research Institute, Bethesda, MD, USA (http://

www.stanleyresearch.org/programs/stanley_research.asp).

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